Enormous efforts are currently being made to develop a vaccine against HIV-1 (Laurence, J. [1990] AIDS Res. 6:175-181). Currently tested systems employ either killed virus, stripped of its coat, or one of the HIV proteins, either surface glycoproteins (gp120, gp160) or core proteins (p24, hgp30). The main limitation with subunit protein vaccines is their poor immunogenicity, even if combined with an adjuvant, e.g., muramyltripeptide or others.
Another approach, used by several investigators, has been to enhance the immunogenic activity of subunit vaccines by preparing protein-containing lipid vesicles, called "proteoliposomes," "immunoliposomes," "immunosomes," "virosomes," or the like. Methods to prepare these vesicles vary widely, however, they are based on one of the following protocols: proteins are usually (i) passively bound to lipids by van der Waal's or hydrophobic interaction; (ii) covalently bound to lipids, or to phospholipid molecules (mainly phosphatidylethanolamine), via divalent coupling agents such as SPDP and others; or (iii) reconstituted with extracted viral lipids, phospholipids, or a combination of both, from detergent-solubilized lipid-protein mixtures.
Only a small number of investigators (Ho, R. J. Y., R. L. Burke, T. C. Merigan [1989] J. Virology 63:2951-2958; El Guink, N., R. M. Kris, G. Goodman-Snitkoff, P. A. Small Jr., and R. J. Mannino [1989] Vaccine 7:147-151; Gould-Fogerite, S., J. E. Mazurkiewicz, D. Bhisitkul, and R. J. Mannino [1988] In Advances in Membrane Biochemistry and Bioenergetics [C. H. Kim et al., eds.] Plenum Press, New York, pp. 569-586; Nussbaum, O., M. Lapidot, and A. Loyter [1987] J. Virol. 61:2245-2252; Haddad, R. S., and L. M. Hutt-Fletcher [1989] J. Virol. 63:4998-5005; Oth, D., G. Mercier, P. Perrin, M. L. Joffret, P. Sureau, and L. Thibodeau [1987] Cell. Immunol. 108:220-226; Thibodeau, L., M. Chagnon, L. Flamand, D. Oth, L. Lachapelle, C. Tremblay, and L. Montagnier [1989] C. R. Acad. Sci. Paris 309(III):741-747) made an effort to insert proteins so that binding and/or immunogenicity was retained upon reconstitution of the mixture, usually via detergent dialysis. However, in most cases, arbitrary phospholipid mixtures are used which do not represent the accurate lipid composition of the viral envelope. Only El Guink et al. (1989, supra) used control piposomes (without viral proteins) similar to the natural viral lipid composition.
Thibodeau et al. (1989, supra) describe a method of "anchoring" HIV gp160 on the surface of liposomes to prepare "HIV-immunosomes." However, the liposome composition is not disclosed, and the "anchoring" is achieved by simple incubation of preformed liposomes with the purified gp160.
In contrast to Thibodeau et al.'s (1989, supra) approach, we have generated a viral envelope identical to the HIV-1 envelope with respect to its lipid composition, an approximately equimolar lipid: cholesterol ratio, unilamellarity, and vesicular size. Furthermore, the incorporation of gp160 was achieved by partial re-solubilization of the lipid envelopes in order to maintain the correct three-dimensional protein conformation by incorporation of the hydrophobic gp41 part of gp160 into the lipid envelope. Using our innovative approach, it is now possible to formulate subunit vaccines which are superior to conventional vaccines.
The subject invention, which utilizes a unique double-detergent dialysis procedure, results in a viral envelope with proteins only on the surface, as they should be, rather than randomly intermingled with the envelope lipid portion.